The acute shortage of human organs for transplantation provides a compelling need for the development of new sources of suitable tissue. An idea of considerable promise is to transplant patents with organs from non-human animals. The main challenge to overcome is rendering foreign tissue immunologically compatible with the patient being treated.
Tissue from most mammalian species would undergo hyperacute rejection when transplanted into humans. This is because human plasma contains natural antibodies against carbohydrate determinants of the animal tissue, thought to originate through prior immune stimulation by dietary antigen or mucosal microflora. Since the antibodies are pre-formed, rejection occurs within days of the transplant.
The main target for the natural antibodies mediating rejection is cell-surface oligosaccharides expressing the determinant Galα(1,3)Gal (reviewed by Joziasse et al., Biochim. Biophys. Acta 1455:403, 1999). Humans, apes and Old World monkeys differ from other mammals in that they lack α-galactosyl epitopes in complex oligosaccharides. Other mammals express the Galα(1,3)Gal epitope prominently on the surface of nucleated cells, including hepatic cells, renal cells, and vascular endothelium—which is especially problematic for xenotransplantation of whole organs.
The Galα(1,3)Gal epitope is made by a specific enzyme, α(1,3) galactosyltransterase, abbreviated in this disclosure as α1,3GT. The transferase uses UDP-galactose as a source of galactose, which it transfers specifically to an acceptor oligosaccharide, usually Galβ(1,4)GlcNAc (N-acetyl lactosamine). In mammals that don't express the Galα(1,3)Gal product, the α1,3GT locus is inactivated (Gailili et al., Proc. Natl. Acad. Sci. USA 15:7401, 1991). There are frameshift and nonsense mutations within the locus, turning it into a non-functional, processed pseudogene (Laarsen et al., J. Biol. Chem. 265:7055, 1990; Joziasse et al., J. Biol. Chem. 266:6991, 1991).
In humans, N-acetyl lactosamine acceptor oligosaccharides are processed differently. The enzyme α(1,2)fucosyltransferase builds the N-acetyl lactosamine into Fucα(1,2)Galβ(1,4)GlcNAc, which is blood group H substance. This in turn serves as an acceptor substance for blood group A GlcNAc-transferase, or blood group B Gal-transferase, forming A-substance or B-substance, respectively, depending on the blood type of the individual. Naturally occurring antibodies circulating in the blood are reactive against the alternative carbohydrate determinants that are not self-antigens.
Larsen et al. (Proc. Nat. Acad. Sci. USA 86:8227, 1989) isolated and characterized a cDNA encoding murine α1,3GT. Joziasse et al. (J. Biol. Chem. 267:5534, 1992) detected four distinct mRNA transcripts, which predict four different isoforms of the α1,3GT. The full-length mouse mRNA (including 5′ untranslated mRNA) was reported to span at least 35-kb of genomic DNA, distributed over nine exons ranging from 36 base pairs to ˜2600 base pairs in length. Numbering in the 5′ to 3′ direction, the coding region is distributed over Exons 4 to 9. The four transcripts are formed by alternative splicing of the pre-mRNA.
Joziasse et al. (J. Biol. Chem. 264:14290, 1989) isolated and characterized a cDNA encoding bovine cDNA. The coding sequence was predicted to be a membrane-bound protein with a large glycosylated COOH-terminal domain, a transmembrane domain, and a short NH2 terminal domain.
The porcine α1,3GT cDNA sequence has been reported from several different laboratories: Strahan et al. (Immunogenetics 41:101, 1995); U.S. Pat. Nos. 5,821,117; 5,849,991; and International Patent Application WO 95/28412. The genomic organization of porcine α1,3GT was reported by Katayama et al. (Glycoconjugate J. 15:83, 1998). Again, the coding region spans six exons, conserving the arrangement present in the mouse genome, and extending over nearly 24-kb.
It has been reported that about 95% of the naturally occurring xenospecific antibody in humans recognize the Galα(1,3)Gal epitope (McKensie et al., Transpl. Immunol. 2:81, 1994). Antibody in human serum binds specifically to pig endothelial cells in a manner that is inhibitable by Galα(1,3)Gal, or by Galα(1, 6)Glc (melibiose). New age monkeys have the same naturally occurring antibody, and demonstrate hyperacute rejection of pig organ xenotransplants. The rejection reaction can be obviated in experimental animals by infusing the recipient with the free carbohydrate (Ye et al., Transplantation 58:330, 1994), or by adsorbing antibody from the circulation on a column of Galα(1,3)Gal or melibiose (Cooper et al., Xenotransplantation 3:102, 1996).
It has been suggested that xenotransplants of pig tissue could provide a source of various tissue components—heart valves, pancreatic islet cells, and perhaps large organs such as livers and kidneys (Cowley, Newsweek, Jan. 1, 2000). If xenotransplants from non-primates into humans is ever to become viable, then techniques need to be developed to prevent Galα(1,3)Gal mediated rejection. Possible genetic manipulation strategies are reviewed by Gustafsson et al. (Immunol. Rev. 141:59, 1994), Sandrin et al. (Frontiers Biosci. 2:e1–11, 1997), and Lavitrano et al. (Forum Genova 9:74, 1999).
One approach is to prevent the formation of Galα(1,3)Gal by providing another transferase that competes with α1,3GT for the N-acetyl lactosamine acceptor. International Patent Application WO 97/12035 (Nextran-Baxter) relates to transgenic animals that express at least one enzyme that masks or reduces the level of the xenoreactive antigens by competing with α1,3GT. The enzymes proposed are α(1,2)fucosyltransferase (that makes H antigen in humans), α(2,6)sialyltransferase, and β(1,3)N-acetylglucosaminyltransferase. It is thought that once N-acetyl lactosamine has been converted by one of these transferases, it can no longer act as an acceptor for α1,3GT. The xenotransplantation cells of Application WO 97/12035 have at least one enzyme that reduces Galα(1,3)Gal expression, and also express a complement inhibitor such as CD59, decay accelerating factor (DAF), or membrane cofactor protein (MCP). Expression of human CD59 in transgenic pig organs enhances organ survival in an ex vivo xenogeneic perfusion model (Kroshus et al., Transplantation 61:1513, 1996).
Another approach is to disassemble Galα(1,3)Gal after it is formed. International Patent Application WO 95/33828 (Diacrin) suggests modifying cells for xenogeneic transplants by treating tissue with an α-glycosidase. Osman et al. (Proc. Natl. Acad. Sci. USA 23:4677, 1997) reported that combined transgenic expression of both α-glycosidase and α(1,2)fucosyltransferase leads to optimal reduction in Galα(1,3)Gal epitope. Splenocytes from mice overexpressing human α-glycosidase showed only a 15–25% reduction in binding of natural human anti-Galα(1,3)Gal antibodies. Mice expressing human α(1,2)fucosyltransferase as a transgene showed a reduction of Galα(1,3)Gal epitopes by ˜90%. Doubly transfected COS cells expressing both the glycosidase and the transferase showed negligible cell surface staining with anti-Galα(1,3)Gal, and were not susceptible to lysis by human serum containing antibody and complement.
A further alternative is to prevent Galα(1,3)Gal expression in the first place. Strahan et al. (Xenotransplantation 2:143, 1995) reported the use of antisense oligonucleotides for inhibiting pig α1,3GT, leading to a partial reduction in expression of the major target for human natural antibodies on pig vascular endothelial cells. Hayashi et al. (Transplant Proc. 29:2213, 1997) reported adenovirus-mediated gene transfer of antisense ribozyme for α1,3GT and α(1,2) fucosyltransferase genes in xenotransplantation.
U.S. Pat. No. 5,849,991 (Bresatch) describes DNA constructs based on the mouse α1,3GT sequence. They are designed to disrupt expression of functional α1,3GT by undergoing homologous recombination across Exon 4, 7, 8, or 9. The constructs contain a selectable marker such as neoR, hygR or thymidine kinase. It is proposed that such constructs be introduced into mouse embryonic stem (ES) cells, and recovering cells in which at least one α1,3GT gene is inactivated. Experiments are reported which produced mice that are homozygous for inactivated α1,3GT, resulting in lack of expression of Galα(1,3)Gal epitope, as determined by specific antibody.
U.S. Pat. No. 5,821,117 (Austin Research Inst.) report cDNA sequence data for porcine α1,3GT. This was used to probe a pig genomic DNA library, and two lambda phage clones were obtained that contain different regions of the porcine transferase gene. International Patent Application WO 95/28412 (Biotransplant) also reports cDNA sequence data for porcine α1,3GT. It is proposed that genomic DNA fragments be isolated from an isogenic DNA library, and used to develop a gene-targeting cassette including a positive or negative selectable marker.
International Patent Application WO 99/21415 (Stem Cell Sciences, Biotransplant) reports construction of a DNA library from miniature swine. A vector is obtained comprising a pgk-neo cassette, and fragments of the α1,3GT gene. This is used for homologous recombination to eliminate α1,3GT activity in porcine embryonic fibroblasts. Costa et al., Alexion Pharmaceuticals (Xenotransplantion 6:6, 1999) report experiments with transgenic mice expressing the human complement inhibitor CD59. In α1,3GT knockout mice, the CD59 gene helped prevent human serum-mediated cytolysis. It had a similar effect in mice expressing α(1,2)fucosyltransferase. Combination of all three modifications provided no additional protective effect.
There have been no reports of the use of α1,3GT inactivated tissue suitable for xenotransplantation into humans. In view of the paucity of available organs for human transplantation, there is a pressing need to develop further options.